Summary To date, targeting the genes regulating intrinsic axon growth ability have produced by far the most promising results in optic nerve regeneration. Recent studies, including ours, have provided strong evidence that neuronal aging might be a key converging process underlying the loss of intrinsic axon growth ability of CNS neurons. Indeed, many genes that act to regulate axon regeneration are also hallmark genes of aging (genomic instability, telomere attrition, epigenetic alteration, and nutrient sensing, etc.). First, recent studies and our preliminary results showed that c-Myc and p53, two well-known genes involved in DNA repair and genomic instability during aging, act to support optic nerve regeneration. Second, our preliminary study showed that telomerase reverse transcriptase (TERT) was necessary for sensory axon regeneration in vivo. Third, aging is often associated with decreased methylation of histone 3 at lysine 27 (H3K27) and increased methylation of H3K4, resulting in reduced amount of heterochromatin. In support, the level of H3K27 demethylase UTX increases during aging and knocking out UTX in c. elegans promotes longevity. Our unpublished study showed that knocking out UTX and its targeted gene, Magi3, in RGCs drastically promoted optic nerve regeneration. Fourth, the insulin and IGF-1 signaling (IIS) pathways, the key regulators of nutrient sensing, are the most conserved aging controlling pathway in evolution. IGF-1 and many IIS downstream targets, such as Pten/PI3K, Akt, and mTor, are all important regulators of optic nerve regeneration. Our published study and a recent study have shown that Sirt1 and LKB1, two important nutrient sensors, function to regulate sensory axon and spinal cord regeneration, respectively. Foxo3, another key target of Akt signaling, has recently been shown to promote vascular cell regeneration through Sirt1. Lastly, recent findings indicated that cellular reprogramming process can reverse aging and rejuvenate the cells. Importantly, manipulations of several reprogramming factors, such as KLF4 and Lin28, have been shown to promote optic nerve regeneration. Therefore, we hypothesize that aging regulatory genes/pathways can be manipulated to promote optic nerve regeneration through rejuvenation of mature CNS neurons. In Aim 1, we will determine if manipulation of miR-138/Sirt1, TERT, and Foxo3 in RGCs can promote optic nerve regeneration. In Aim 2, we will first determine if combination of these aging genes with myosin II knockout or enhanced neural activity would have synergistic effects on regeneration. We will then use RNA-seq and ATAC-seq of purified RGCs to explore how these aging genes regulate optic nerve regeneration. In Aim 3, by performing RNA-seq and ATAC-seq of purified RGCs at different developing, maturation, and aging stages, we will first use advanced integrative bioinformatics analyses to identify top candidate aging genes and their associated transcription factors, both of which act to orchestrate RGCs aging. We will then perform functional screening experiments to determine their roles in regulation of axon growth and optic nerve regeneration.